For intracellular FRET, THP-1 cells were incubated with 1 μM FITC-conjugated IRF5-CPPs for 1 hour, fixed, permeabilized, and stained with anti-IRF3 (Abcam, ab76409), anti-IRF5 (ab124792; or Cell Signaling Technology, cs13496), anti-IRF7 (cs4920), or anti–NF-κB (cs8242) antibodies and TRITC-conjugated secondary antibodies (Abcam). Cell-associated fluorescence was measured on BioTek Synergy Neo2 (BioTek, VT) at 525 nm upon excitation at 488 nm (E1), at 600 nm after excitation at 540 nm (E2), and at 600 nm after excitation at 488 nm (E3). The transfer of fluorescence was calculated as FRET units as follows: FRET unit = (E3bothE3none) − ([E3TRITCE3none) × (E2both/E2TRITC]) − ([E3FITCE3none] × [E1both/E1FITC]) (53). The different fluorescence values (E) were measured on unlabeled cells (Enone) or cells labeled with FITC (EFITC) and TRITC (ETRITC). In a similar manner, FRET signal was analyzed by imaging flow cytometry on an ImageStreamX Mark II (EMD Millipore), and FITC-TRITC similarity scores were determined using IDEAS software package (55).

IRF3, IRF5, and IRF7 homodimerization were determined by Native gel electrophoresis after preincubation of THP-1 monocytes with IRF5-CPPs (1 and 10 μM) and stimulation with R848 for 1 hour. An 8% Native gel was prerun in 25 mM tris/192 mM glycine (pH 8.3) with 1% (w/v) deoxycholate in the cathode chamber for 30 min at 40 mA at 0°C. Cell lysates (10 μg) were mixed with Native sample buffer [62.5 mM tris-Cl (pH 6.8), 15% glycerol, and 1% bromophenol blue) and electrophoresed for 60 min at 25 mA and 0°C. The gel was then incubated for 30 min with 25 mM tris/192 mM glycine (pH 8.3) and 0.1% SDS before protein transfer. Homodimer and monomer IRFs and β-actin were detected with anti-IRF3 (ab76409), anti-IRF5 (cs3257 or cs13496), or anti-IRF7 (cs4920) and horseradish peroxidase (HRP)–conjugated monoclonal anti–β-actin (cs5125) antibodies, respectively.

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